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Updated: Feb 15, 2026
Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
Published on: October 22, 2012
Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells
Binoy Appukuttan1, Yuefang Ma1, Andrew Stempel1
1Eye and Vision Health, College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia.
Background:
Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans.
Methods:
Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production.
Results:
Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth.
Conclusions:
Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.
Insights
Reactive oxygen species (ROS) cause retinal damage. Inhibiting Nox4 enzymes with GKT136901 or GKT137831 reduced ROS and protected human retinal cells, supporting their use in treating vascular diseases.
Area of Science:
- Ophthalmology
- Vascular Biology
- Biochemistry
Background:
- Oxidative stress from reactive oxygen species (ROS) contributes to retinal damage in vascular diseases like diabetic retinopathy.
- NADPH oxidase (Nox) enzymes are key ROS producers, making them attractive therapeutic targets.
- Previous studies show Nox4 inhibition lessens experimental retinal vasculopathy.
Purpose of the Study:
- To investigate the human relevance of Nox4 inhibition for retinal vascular diseases.
- To assess the efficacy of Nox1/Nox4 inhibitors (GKT136901, GKT137831) in human retinal endothelial cells.
Main Methods:
- Human retinal endothelial cells were treated with dimethyloxalylglycine (DMOG) to induce oxidative stress.
- Nox4 expression, ROS production, and vascular endothelial growth factor A (VEGFA) transcript levels were measured.
- Cells were then treated with Nox1/Nox4 inhibitors GKT136901 or GKT137831.
Main Results:
- Nox4 was the predominant Nox enzyme in human retinal endothelial cells.
- DMOG increased Nox4 expression, ROS production, and VEGFA levels.
- GKT136901 and GKT137831 significantly reduced DMOG-induced ROS, VEGFA expression, and inhibited DMOG's effect on cell growth.
Conclusions:
- Findings in human retinal endothelial cells translate experimental model results.
- Nox4 inhibition using GKT136901 and GKT137831 shows therapeutic potential for patients with retinal vascular diseases.
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